Rho kinase inhibitors for use in treating familial amyotrophic lateral sclerosis

ABSTRACT

The invention relates to a new use of a known Rho kinase inhibitor, fasudil or a fasudil derivative selected from hydroy-fasudil or dimethylfasudil, in the treatment of amyotrophic lateral sclerosis (ALS).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the National Phase of PCT International ApplicationNo. PCT/EP2013/054804, filed on Mar. 11, 2013, which claims priorityunder 35 U.S.C. 119(e) to U.S. Provisional Application No. 61/609,576,filed on Mar. 12, 2012, all of which are hereby expressly incorporatedby reference into the present application.

The present invention relates to a new use of a known Rho kinaseinhibitor in the treatment of amyotrophic lateral sclerosis (ALS).

BACKGROUND OF THE INVENTION

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease,is one of the most common neuromuscular diseases worldwide, and peopleof all races and ethnic backgrounds are affected. ALS is most commonlydiagnosed in people between 40 and 60 years of age, and men are affectedslightly more often than women. One or two out of 100,000 people developALS each year and estimated 5,000 people in the United States arediagnosed with the disease each year. ALS is a progressive diseasecharacterized by rapidly progressive weakness, muscle atrophy andfasciculation, spasticity, dysarthria, dysphagia, and respiratorycompromise. It is a neurodegenerative movement disorder caused by thedegeneration of neurons located in the ventral horn of the spinal cordand the cortical neurons. Unable to function, the muscles weaken andatrophy. Cognitive function is generally spared for most patientsalthough ˜5% also develop frontotemporal dementia. ˜30-50% of patientsalso show subtle cognitive changes which can be revealed by detailedneuropsychological testing.

There is a known hereditary factor in familial ALS, although thisaccounts for only around 5-10% of all cases. Approximately 20% offamilial cases of ALS have been linked with an autosomal dominantgenetic defect on chromosome 21, coding for superoxide dismutase (SOD1).The most common ALS causing SOD1 mutation in North America is A4V,characterized by an exceptionally rapid progression from onset to death.A recent study identified a gene called FUS (“Fused in Sarcoma”, ALS6)as being responsible for 1 in 20 cases of familial ALS.

The degeneration of the first and second motoneuron in ALS is amultifactorial pathologic process. A variety of neuroprotective agentshave been examined to counteract initiation and/or progression ofdisease, but only the glutamate antagonist Riluzole (marketed bySanofi-Aventis under the trademark Rilutek), which targets glutamatetransporters, has reached clinical use so far. It has been found toimprove survival to a modest extent and also extends the time before aperson needs ventilation support. Riluzole does not reverse the damagealready done to motor neurons, and people taking it must be monitoredfor liver damage (occurring in ˜10% of people taking the drug). Furtherknown side-effects or Riluzole are nausea and fatigue which may causepatients to discontinue treatment.

Fasudil is a potent Rho-kinase (ROCK) inhibitor and vasodilator. In thepast, it has been used for the treatment of cerebral vasospasm, which isoften due to subarachnoid hemorrhage, as well as to improve thecognitive decline seen in stroke victims. Moreover, it has been found tobe useful for the treatment of pulmonary hypertension and to enhancememory and improve the prognosis of Alzheimer patients.

The inventors have previously shown that pharmacological inhibition ofrho kinase by Y-27632 (a 4-aminopyridine derivative) and Fasudil (anisoquinoline derivative) does not only enhance regeneration, but alsosurvival of lesioned CNS neurons in vivo and in vitro (Lingor, Teusch etal. 2007; Lingor, Tonges et al. 2008; Planchamp, Bermel et al. 2008;Bermel, Tonges et al. 2009). In addition to the established effects onthe actin cytoskeleton, the inventors could show that inhibition of ROCKresults in activation of intracellular pathways mediating neuronalsurvival. Further, the inventors have demonstrated that pharmacologicROCK inhibition in the optic nerve lesion paradigm leads to aregeneration of lesion optic nerve axons and is able to improve survivalof lesioned retinal ganglion cells (Lingor et al. 2010). Theneuroprotective potential of ROCK inhibition in neurodegenerativedisease has been further corroborated by other groups. For example, animprovement of neurological function and an increase of survival inanimal models of Huntington's disease and SMA was reported (Li, Huang etal. 2009; Bowerman, Beauvais et al. 2010). However, a distinct survivalprolonging effect in these animals models was only shown in rather acuteneurodegenerative models like in the optic nerve lesion paradigm whereretinal ganglion cell survival is evaluated 14 days after lesion and inthe SMA model in which mice usually die after several weeks of life.

WO 2009/155777 A1 relates to the application of Fasudil in inducingregeneration of stem cells of adult cranial nerves. WO 2005/117896 A1discloses pharmaceutical formulations of Fasudil and their use in thetreatment of inter alia neuronal regeneration.

There is still a need in the art for new and effective treatments ofALS, in particular for treatments having less side-effects than thecurrently known ones.

SUMMARY OF THE INVENTION

The inventors decided to evaluate the therapeutic potential of the ROCKinhibitor Fasudil in the chronic SOD1 G93A mouse model of ALS((B6SJL-TgN (SOD1-G93A) 1 Gur), that reflects the evolution of ALSdisease clinically very well over a period of up to 22 weeks. The studywas designed to comprise a presymptomatic (d50) and a symptomatic (d80)pharmacologic treatment group with Fasudil versus vehicle control.Parameters to be evaluated were disease onset, prolongation of survivaland motor function. Furthermore, the inventors performed a detailedanalysis of the influence of the treatment on ALS pathology in thecentral and peripheral nervous system thus taking into account allprincipally affected compartments of this neurodegenerative disease. Inparticular, the inventors undertook an extensive immunohistologicexamination of the different treatment groups and evaluated as wellsurvival of spinal cord motoneurons as glial infiltration withastrocytes and activated microglia in the spinal cord.

The data shown in the Examples demonstrates that treatment with Fas30significantly prolongs survival of female SOD1 G93A mice. Thisbeneficial effect is also reflected in an improved motor coordinationfunction as demonstrated in the rotarod test and is mirroredimmunopathologically in an increased survival of spinal cord motoneuronsand a decreased astrogliotic infiltration of the spinal cord.

Accordingly, in a first aspect, the invention relates to apharmaceutical composition, comprising1-(5-isoquinolinesulfonyl)homopiperazine (fasudil), or apharmaceutically acceptable salt thereof, for use in the treatment of asubject suffering from ALS. Preferably, the pharmaceutical compositionconsists of fasudil. In one embodiment, the ALS is sporadic ALS. Inanother embodiment, the ALS is familial ALS.

Likewise, the invention relates to a pharmaceutical composition,comprising 1-(5-isoquinolinesulfonyl)homopiperazine (fasudil), or apharmaceutically acceptable salt thereof, for use in the treatment of asubject prone to suffer from ALS. Preferably, the pharmaceuticalcomposition consists of fasudil. In one embodiment, the ALS is sporadicALS. In a more preferred embodiment, the ALS is familial ALS.

The invention further relates to a pharmaceutical composition,comprising a fasudil derivative, or a pharmaceutically acceptable saltthereof, for use in the treatment of a subject suffering from ALS.Preferably, the pharmaceutical composition consists of said fasudilderivative. In one embodiment, the ALS is sporadic ALS. In anotherembodiment, the ALS is familial ALS. In one embodiment, the fasudilderivative is 1-(6-hydroxyl-5-isoquinolinesulfonyl)homopiperazine(hydroxyl-fasudil). In another embodiment, the fasudil derivative is(S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine(dimethyl-fasudil).

Likewise, the inventions relates to a pharmaceutical composition,comprising a fasudil derivative, or a pharmaceutically acceptable saltthereof, for use in the treatment of a subject prone to suffer from ALS.Preferably, the pharmaceutical composition consists of said fasudilderivative. In one embodiment, the ALS is sporadic ALS. In a morepreferred embodiment, the ALS is familial ALS. In one embodiment, thefasudil derivative is1-(6-hydroxyl-5-isoquinolinesulfonyl)homopiperazine (hydroxyl-fasudil).In another embodiment, the fasudil derivative is(S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine(dimethyl-fasudil).

Thus, the invention relates to a pharmaceutical composition, comprisingor consisting of fasudil, or a fasudil derivative selected fromhydroxyl-fasudil and dimethyl-fasudil, or a pharmaceutically acceptablesalt thereof, for use in the treatment of a subject suffering or proneto suffer from sporadic or familial ALS.

In other words, a method of treating a subject suffering or prone tosuffer from sporadic or familial ALS is provided, wherein the methodcomprises administering a pharmaceutical composition, comprising orconsisting of fasudil, or a fasudil derivative selected fromhydroxy-fasudil and dimethyl-fasudil, or a pharmaceutically acceptablesalt thereof, to the subject.

In another aspect, the invention provides a pharmaceutical composition,comprising fasudil, or a pharmaceutically acceptable salt thereof, foruse of improving motor coordination in a subject suffering from ALS.Preferably, the pharmaceutical composition consists of fasudil. In oneembodiment, the ALS is sporadic ALS. In another embodiment, the ALS isfamilial ALS.

Likewise, the invention relates to a pharmaceutical composition,comprising fasudil, or a pharmaceutically acceptable salt thereof, foruse of improving motor coordination in a subject prone to suffer fromALS. Preferably, the pharmaceutical composition consists of fasudil. Inone embodiment, the ALS is sporadic ALS. In a more preferred embodiment,the ALS is familial ALS.

The invention further provides a pharmaceutical composition, comprisinga fasudil derivative, or a pharmaceutically acceptable salt thereof, foruse of improving motor coordination in a subject suffering from ALS.Preferably, the pharmaceutical composition consists of said fasudilderivative. In one embodiment, the ALS is sporadic ALS. In anotherembodiment, the ALS is familial ALS. In one embodiment, the fasudilderivative is 1-(6-hydroxyl-5-isoquinolinesulfonyl)homopiperazine(hydroxyl-fasudil). In another embodiment, the fasudil derivative is(S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine(dimethyl-fasudil).

Likewise, the invention relates to a pharmaceutical composition,comprising a fasudil derivative, or a pharmaceutically acceptable saltthereof, for use of improving motor coordination in a subject prone tosuffer from ALS. Preferably, the pharmaceutical composition consists ofsaid fasudil derivative. In one embodiment, the ALS is sporadic ALS. Ina more preferred embodiment, the ALS is familial ALS. In one embodiment,the fasudil derivative is1-(6-hydroxyl-5-isoquinolinesulfonyl)homopiperazine (hydroxyl-fasudil).In another embodiment, the fasudil derivative is(S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine(dimethyl-fasudil).

Thus, the invention relates to a pharmaceutical composition, comprisingor consisting of 1-(5-isoquinolinesulfonyl)homopiperazine (fasudil), ora fasudil derivative selected from1-(6-hydroxyl-5-isoquinolinesulfonyl)homopiperazine (hydroxyl-fasudil)and(S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine(dimethyl-fasudil), or a pharmaceutically acceptable salt thereof, foruse of improving motor coordination in a subject suffering or prone tosuffer from sporadic or familial ALS.

In other words, the invention provides a method of improving motorcoordination in a subject suffering or prone to suffer from sporadic orfamilial ALS, wherein the method comprises administering apharmaceutical composition, comprising or consisting of fasudil, or afasudil derivative selected from hydroxy-fasudil and dimethyl-fasudil,or a pharmaceutically acceptable salt thereof, to the subject. Forexample, an improvement of the motor coordination in a subject can bedetermined in accordance with the ALS functional rating scale.

Finally, the invention provides a method of prolonging lifetime of asubject suffering from ALS, wherein the method comprises administering apharmaceutical composition, comprising fasudil, or a pharmaceuticallyacceptable salt thereof, to the subject. In one embodiment, thepharmaceutical composition consists of fasudil. The ALS may be sporadicALS. Alternatively, the ALS may be familial ALS. Likewise, the inventionprovides a method of prolonging lifetime of a subject suffering fromALS, wherein the method comprises administering a pharmaceuticalcomposition, comprising a fasudil derivative, or a pharmaceuticallyacceptable salt thereof, to the subject. In one embodiment, thepharmaceutical composition consists of said fasudil derivative. In oneembodiment, the fasudil derivative is hydroxyl-fasudil. In anotherembodiment, the fasudil derivative is dimethyl-fasudil. The ALS may besporadic ALS. Alternatively, the ALS may be familial ALS.

Also provided is a method of prolonging lifetime of a subject prone tosuffer from ALS, wherein the method comprises administering apharmaceutical composition, comprising fasudil, or a pharmaceuticallyacceptable salt thereof, to the subject. In one embodiment, thepharmaceutical composition consists of fasudil. Alternatively, themethod comprises administering a pharmaceutical composition, comprisinga fasudil derivative, or a pharmaceutically acceptable salt thereof, tothe subject. In one embodiment, the pharmaceutical composition consistsof said fasudil derivative. In one embodiment, the fasudil derivative ishydroxyl-fasudil. In another embodiment, the fasudil derivative isdimethyl-fasudil. The ALS may be sporadic ALS. Preferably, the ALS maybe familial ALS.

Hence, the invention provides a method of prolonging lifetime of asubject suffering or prone to suffer from sporadic or familial ALS,wherein the method comprises administering a pharmaceutical composition,comprising or consisting of fasudil, or a fasudil derivative selectedfrom hydroxy-fasudil and dimethyl-fasudil, or a pharmaceuticallyacceptable salt thereof, to the subject.

With regard to any aspect described herein, the ALS, in particular thesporadic or familial ALS, is early stage ALS as diagnosable by theAwaji-shima diagnostic criteria. It is further preferred that theadministration of said composition is started presymptomatically. Thepharmaceutical composition is particularly effective if the subject is afemale. Preferably, the pharmaceutical composition is to be administeredorally. It is further preferred that the fasudil is to be administeredto the subject in a dosage of 1-12 mg/kg body weight per day, preferablyin a dosage of 1.25-8 mg/kg body weight per day, such as in a dosage of1.5-6 mg/kg body weight per day, more preferably in a dosage of 1.75-5mg/kg body weight per day, even more preferably in a dosage of 2-4 mg/kgbody weight per day, and most preferably in a dosage of 2.4-3.6 mg/kgbody weight per day. In contrast thereto, the fasudil derivative ispreferably to be administered to the subject in a dosage of 10-1200ng/kg body weight per day, preferably in a dosage of 12.5-800 ng/kg bodyweight per day, such as in a dosage of 15-600 ng/kg body weight per day,more preferably in a dosage of 17.5-500 ng/kg body weight per day, evenmore preferably in a dosage of 20-400 ng/kg body weight per day, andmost preferably in a dosage of 24-360 ng/kg body weight per day.Preferably, the pharmaceutical composition is formulated as aformulation with sustained release or prolonged release. For example, animprovement of the motor coordination in a subject can be determined inaccordance with the ALS functional rating scale.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In a first aspect, the invention relates to a pharmaceuticalcomposition, comprising or consisting of1-(5-isoquinolinesulfonyl)homopiperazine (fasudil), or a fasudilderivative selected from1-(6-hydroxyl-5-isoquinolinesulfonyl)homopiperazine (hydroxyl-fasudil)and(S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine(dimethyl-fasudil), or a pharmaceutically acceptable salt thereof, foruse in the treatment of a subject suffering or prone to suffer fromsporadic or familial ALS. Likewise, the present invention provides amethod of treating a subject suffering or prone to suffer from sporadicor familial ALS, wherein the method comprises administering apharmaceutical composition, comprising or consisting of fasudil, or afasudil derivative selected from hydroxy-fasudil and dimethyl-fasudil,or a pharmaceutically acceptable salt thereof, to the subject.

Fasudil, hydroxyl-fasudil and dimethyl-fasudil are known in the art, anddescribed, e.g., in U.S. Pat. No. 4,678,783. Fasudil and the fasudilderivative may be used in their free forms or as salts thereof. The saltis a non-toxic salt which is pharmacologically accepted and may beformed by addition of an acid. As examples of the acid, there may bementioned such inorganic acids as hydrochloric acid, hydrobromic acid,phosphoric acid and sulfuric acid and such organic acids as acetic acid,citric acid, tartaric acid, lactic acid, succinic acid, fumaric acid,maleic acid and methanesulfonic acid. Preferably, the salt is ahydrochloride, hydrochloride hemihydrate, hydrochloride hydrate,hydrochloride trihydrate, phosphate monobase, phosphate dibase,phosphate, hydrogen sulfate, sulfate, mesylate, ethylsulfonate, maleate,fumarate, or tartrate. More preferably, the fasudil or fasudilderivative is in form of a hydrochloride, hydrochloride hemihydrates orhydrochloride hydrate, as, e.g., described in EP 1 110 553 and U.S. Pat.No. 5,942,505, both which are incorporated herewith by reference.Moreover, fasudil and its derivatives may be used in form of theiractive metabolites formed in vivo. Such active metabolites are known inthe art. Most preferably, fasudil or its derivative is in the form of ahydrochloride. The pharmaceutical composition may comprise fasudil, afasudil derivative, or a pharmaceutically acceptable salt thereof.Alternatively, the pharmaceutical composition may consist of fasudil, afasudil derivative, or a pharmaceutically acceptable salt thereof as theonly active agent. However, the pharmaceutical composition may comprisefurther auxiliary agents, such as fillers, solvents and stabilizers suchas preservatives, as well as suspending and/or dispersing agents.

Administration of the pharmaceutical composition may be effected oradministered by different ways, e.g., enterally, orally (e.g., pill,tablet (buccal, sublingual, orally, disintegrating, capsule, thin film,liquid solution or suspension, powder, solid crystals or liquid),rectally (e.g., suppository, enema), via injection (e.g., intravenously,subcutaneously, intramuscularly, intraperitoneally, intradermally) viainhalation (e.g., intrabronchially), topically, vaginally,epicutaneously, or intranasally. In one embodiment, the composition isadministered via injection, in particular intravenously, subcutaneously,intramuscularly, intraperitoneally, or intradermally; more particularwherein the composition is administered intravenously. Accordingly, thepharmaceutical composition may be in the form of an injectable solution,suppository, nasal spray, time-release implant, transdermal patch, orthe like. Preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

The injectable formulation may be presented in unit dosage form, e.g.,in ampules or in multidose containers, or in powder form forreconstitution with a suitable vehicle, including but not limited tosterile pyrogen free water, buffer, ringer solution or dextrose solutionbefore use. For transdermal administration, the pharmaceuticalformulation may be in the form of an adhesive disc or patch, whichslowly releases the active ingredient for percutaneous absorption.Optionally, such pharmaceutical formulations for transdermal deliverymay be formulated along with permeation enhancers, which may be used tofacilitate transdermal penetration. For rectal and vaginal routes ofadministration, the active ingredients may be formulated as ointments,solutions, or suppositories. For buccal administration, thepharmaceutical composition may take the form of tablets or lozengesformulated in conventional manner. However, the pharmaceuticalcomposition may also be formulated for administration via inhalation,for example in form of a pressurized pack or nebulizer. In case of apressurized aerosol, the dosage unit may be determined by a metereddosage unit, using a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, or carbon dioxide.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the activeagent and a suitable powder base such as lactose or starch. However,most preferably, the pharmaceutical composition is administered orally,for example, in form of a tablet or capsule. The pharmaceuticalcomposition for oral administration may be formulated withpharmaceutically acceptable excipients such as binding agents (e.g.,pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talcor silica); disintegrants (e.g., potato starch or sodium starchglycolate); or wetting agents (e.g., sodium lauryl sulfate), and may becoated by methods well known in the art. Liquid preparations for oraladministration such as solutions, syrups or suspensions, may be preparedwith pharmaceutically acceptable additives such as suspending agents(e.g., sorbitol syrup, cellulose derivatives or hydrogenated ediblefats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts, flavoring, coloring and sweetening agents.

Preferably, the fasudil, fasudil derivative, or a pharmaceuticallyacceptable salt thereof is formulated as a formulation with sustainedrelease or prolonged release in order to provide extended plasmahalf-life and avoid variations in plasma concentration between dosingand eventually enhance bioavailability of the drug. For prolongeddelivery, the pharmaceutical composition may be formulated as a depotpreparation, for administration by implantation; e.g., subcutaneous,intradermal, or intramuscular injection. Thus, for example, thepharmaceutical composition may be formulated with suitable polymeric orhydrophobic materials (e.g., as an emulsion in an acceptable oil) or assparingly soluble derivatives; e.g., as a sparingly soluble salt.Formulations with sustained release or prolonged release of fasudil or afasudil derivative, in particular for oral administration, are known inthe art, e.g. from WO 2005/117896, which is incorporated herewith byreference. The pharmaceutical composition may, be provided in a pack,such as a blister pack, which may contain one or more unit dosage forms,and which may be accompanied by instructions for administration.

The subject may be a mammal, such as a mouse, rat, guinea pig, dog, cat,pig, sheep, horse, or cow. Preferably, the subject is a primate, andmost preferably a human. As it can be taken from the experimentalsection, the invention is preferably effective in females. Accordingly,in a preferred embodiment of the invention, the subject is a female.

In one embodiment, the ALS is sporadic ALS. As derivable from the term“sporadic”, said type of ALS is rare and scattered in occurrence andthus patternless. However, the ALS may also be familial ALS, wherein thesubject is genetically predisposed to develop ALS. The following geneshave been linked with the development of ALS: Autosomal dominant: ALS-1,Ch 21q, SOD1; ALS-2, Ch 18q21; ALS-4, Ch 9q34, Senataxin; ALS-6, Ch16p11, FUS; ALS-7, Ch 20p; ALS-9, Ch 20q13, VAPB; ALS-9, Ch 14q11,Angiogenin; ALS-10, Ch 1p36, TARDBP; ALS-11, Ch 6q21, FIG4 mutations;ALS-12, Ch 10p15, Optineurin variants or mutations; Ch 12q24, D-Aminoacid oxidase mutation; Ch 9p13, VCP mutations; ALS-FTLD, Ch 9q21,Unknown gene. Autosomal recessive: ALS-2, Ch 2q33, Alsin, ALS-5, Ch15q21, Spatacsin; ALS-12, 10p15, Optineurin. For a review, see deCarvalho & Swash (2011), which is incorporated herewith by reference.Subjects who are suspected to have or develop ALS are commonly referredto herein as subjects prone to suffer from ALS. As shown in theexamples, it is advantageous to start treatment of ALS in accordancewith the present invention as early as possible. Therefore, thetreatment using the pharmaceutical composition according to theinvention is preferably started presymptomatically. This may be the caseif the subject is suspected to have ALS, although no final conclusioncould be drawn, or if the subject is prone to suffer from ALS.

ALS is difficult to diagnose, although certain biomarkers have beenreported to correlate with ALS (cf. Carvalho & Swash (2011); sectionheaded “Biomarkers in amyotrophic lateral sclerosis”). ALS causes muscleweakness and atrophy throughout the body caused by degeneration of theupper and lower motor neurons. The earliest symptoms of ALS aretypically obvious weakness and/or muscle atrophy. Other presentingsymptoms include muscle fasciculation (twitching), cramping, orstiffness of affected muscles; muscle weakness affecting an arm or aleg; and/or slurred and nasal speech. The parts of the body affected byearly symptoms of ALS depend on which motor neurons in the body aredamaged first. Patients with the leg onset form may experienceawkwardness when walking or running or notice that they are tripping orstumbling, often with a “dropped foot” which drags gently along theground. Arm-onset patients may experience difficulty with tasksrequiring manual dexterity such as buttoning a shirt, writing, orturning a key in a lock. Indeed, the presence of upper and lower motorneuron signs in a single limb is strongly suggestive of ALS.Electromyography (EMG) is a special technique that detects electricalactivity in muscles, and certain EMG findings can support the diagnosisof ALS.

The use of electrophysiological data in ALS diagnosis was reviewed at aconsensus conference held in Awaji-shima, Japan in 2006. It wasrecommended that electrophysiological findings should be taken asequivalent to clinical assessment in the recognition of lower motorneuron involvement. Further, in the context of a suspected clinicaldiagnosis of ALS, fasciculation potentials (FPs) should be taken asequivalent to fibrillation potentials (fibs) and positive sharp waves(sw) in recognizing denervation, in particular in strong limb musclesand in cranial-innervated muscles. Finally, the importance of searchingfor instability in fasciculation potentials and in motor unit potentials(MUP) in ALS was stressed. The earlier topographic criteria set out inthe El Escorial criteria were unchanged. The Awaji-shima consensusrecommendations for the application of electrophysiological tests to thediagnosis of ALS are as follows:

1. Principles (from the Airlie House Criteria)

-   -   The diagnosis of amyotrophic lateral sclerosis [ALS] requires    -   (A) the presence of (1) evidence of lower motor neuron (LMN)        degeneration by clinical, electrophysiological or        neuropathological examination; (2) evidence of upper motor        neuron (UMN) degeneration by clinical examination; and (3)        progressive spread of symptoms or signs within a region or to        other regions, as determined by history, physical examination,        or electrophysiological tests;    -   (B) the absence of (1) electrophysiological or pathological        evidence of other disease processes that might explain the signs        of LMN and/or UMN degeneration, and (2) neuroimaging evidence of        other disease processes that might explain the observed clinical        and electrophysiological signs.        2. Diagnostic Criteria    -   Clinically definite ALS is defined by clinical or        electrophysiological evidence by the presence of LMN and/or UMN        signs in the bulbar region and at least two spinal regions or        the presence of LMN and UMN signs in three spinal regions.    -   Clinically probable ALS is defined on clinical or        electrophysiological evidence by LMN and UMN signs in at least        two regions with some UMN signs necessarily rostral to (above)        the LMN signs.    -   Clinically possible ALS is defined when clinical or        electrophysiological signs of UMN and LMN dysfunction are found        in only one region; or UMN signs are found alone in two or more        regions; or LMN signs are found rostral to UMN signs.        Neuroimaging and clinical laboratory studies will have been        performed and other diagnoses must have been excluded.

The criteria for detection of neurogenic change by needleelectromyography (EMG) in the diagnosis of ALS are as follows:

-   1. For the evaluation of LMN disease in ALS in any given body region    clinical and electrophysiological abnormalities have equal    diagnostic significance.-   2. EMG features of chronic neurogenic change must be found, for    example    -   a. MUPs of increased amplitude and increased duration, usually        with an increased number of phases, as assessed by qualitative        or quantitative studies.    -   b. Decreased motor unit recruitment, defined by rapid firing of        a reduced number of motor units. In limbs affected by clinical        features of significant UMN abnormalities, rapid firing may not        be achieved.    -   c. Using a narrow band pass filter (500 Hz to 5 kHz) unstable        and complex MUPs will be observed in most cases of ALS.-   3. In ALS fbs-sw are usually recorded in strong, non-wasted,    muscles.-   4. In the presence of chronic neurogenic change on needle EMG in    ALS, fasciculation potentials (FPs), preferably of complex    morphology, are equivalent to fibrillations and positive sharp waves    (fibs-sw) in their clinical significance.

Complex FPs: one feature of polyphasic (>4 phases), increased duration,or increased amplitude compared to normal values for MUPs in the musclestudied. Complex FPs may be unstable.

Fibs-sw: (AANEM definition) duration<5 ms, amplitude<1 mV, dischargefrequency 1-50 Hz.

Unstable MUPs: use low band pass filter setting >500 Hz; superimposesequence of consecutive motor unit discharges, and look for increasedjitter (jiggle) and impulse blocking.

Chronic neurogenic change: MUPs of increased duration, increasedamplitude, and often with increased phases. A decreased interferencepattern characterized by increasing firing rate of remaining motorunits, and increased envelope amplitude of the interferential pattern.

Other disorders may be excluded by nerve conduction studies, wherein thefollowing are compatible with ALS:

-   -   Normal SNAP amplitude and sensory conduction velocities (CV) in        the absence of concomitant entrapment or other neuropathies.        Mildly reduced SNAP amplitudes and CVs in the presence of        neuropathy of identified aetiology are acceptable.    -   Motor CV >75% of the lower limit of normal, and minimum F-wave        latency <130% of the upper limit of normal.    -   Distal CMAP latency and duration <150% of normal.    -   Absence of conduction block (CB) and of pathological temporal        dispersion, as defined by baseline-negative CMAP area reduction        on proximal versus distal stimulation >50% when distal        baseline-negative peak CMAP amplitude is large enough to allow        such assessment (usually >1 mV). A proximal negative peak CMAP        duration <30% of the distal value suggests CB.

The utility of the Awaji-shima revision of the Airlie House diagnosticcriteria has been proven successful in several studies, wherein it wasfound that the diagnostic sensitivity improved when the Awaji-shimacriteria were applied, without loss of specificity. Utilizing theAwaji-shima criteria allows an earlier diagnosis of ALS, and thus anearlier treatment of ALS. For a review, see de Carvalho et al. (2008),which is incorporated herewith by reference. The skilled in the artknows how to perform these tests. As shown in the experimental section,treatment with fasudil and/or a fasudil derivative appears to be mosteffective if the ALS is in an early stage, that is before or when firstelectrophysiological signs, which are predictive of ALS, are detectable.Hence, the present invention is particularly useful if the subject isdiagnosed for suspected ALS or possible ALS. The invention is furtherparticularly useful, if the sporadic or familial ALS is early stage ALSas diagnosable by the Awaji-shima diagnostic criteria, in particular asdetectable by needle EMG, more particular as detectable by EMG featuresof chronic neurogenic change as defined in point 2 above, mostpreferably by EMG features of chronic neurogenic change as defined inpoint 2a, point 2b, point 2c, point 2a and 2b, point 2a and 2c, point 2band 2c, or point 2a and 2b and 2c. Although it might be helpful, in thecontext of the present invention it is not absolutely necessary toexclude other disorders.

In the experimental section, the inventors monitored the drinkingamounts of mice that were determined with 0.2 ml/g body weight/day priorto treatment initiation. To reach sufficient CSF levels of Fasudil theinventors administered Fasudil (LC Labs, Woburn, USA; product numberF-4660) at a low concentration of 15 mg (Fas30) or at a highconcentration of 50 mg (Fas100) in 100 ml drinking water. Control groupsreceived the respective amount of isotonic drinking water termed asvehicle (Veh). Accordingly, the mice received a dosage of 0.2 ml/g bodyweight/day×(15 mg/100 ml)=0.03 mg/g body weight=30 mg/kg body weight(Fas30); and 0.2 ml/g body weight/day×(50 mg/100 ml)=100 mg/kg bodyweight (Fas100), respectively.

Dose translation from one animal to another animal or from an animal tohuman studies is well known in the art, cf. Reagan-Shaw et al. (2007),which is incorporated herewith by reference, in particular FIG. 1 andTable 1. Accordingly, the human equivalent dose (HED; mg per kg bodyweight) may be calculated as follows: HED (mg/kg)=Animal dose(mg/kg)×(Animal Km/Human Km). The Km factor, body weight (kg) divided bybody surface area (BSA; m²), is used to convert the mg/kg dose used in astudy to an mg/m² dose. Exemplary Km values based on average BSAcalculations on data from FDA Draft Guidelines are given below.

Species Weight (kg) BSA (m²) Km factor Human Adult 60 1.6 37 Child 200.8 25 Baboon 12 0.6 20 Dog 10 0.5 20 Monkey 3 0.24 12 Rabbit 1.8 0.1512 Guinea Pig 0.4 0.05 8 Rat 0.15 0.025 6 Hamster 0.08 0.02 5 Mouse 0.020.007 3

In view of the experimental data given below, if the pharmaceuticalcomposition comprises or consists of fasudil or a pharmaceuticallyacceptable salt thereof, the fasudil is to be administered to thesubject in a dosage of 1-12 mg/kg body weight per day, preferably in adosage of 1.25-8 mg/kg body weight per day. Nevertheless, it could bedemonstrated that the Fas30 treatment was particularly effective.Accordingly, in a preferred embodiment, fasudil is to be administered ina dosage of 1.5-6 mg/kg body weight per day, preferably in a dosage of1.75-5 mg/kg body weight per day, more preferably in a dosage of 2-4mg/kg body weight per day, and most preferably in a dosage of 2.4-3.6mg/kg body weight per day.

Alternatively, the pharmaceutical composition may comprise or consist ofa fasudil derivative, in particular selected from hydroxy-fasudil anddimethyl-fasudil, or a pharmaceutically acceptable salt thereof. It isknown in the art that the effect of fasudil derivatives such ashydroxy-fasudil and dimethyl-fasudil is 10 to 100 times higher ascompared to fasudil. Based on the data for fasudil, it is thus preferredthat the fasudil derivative is administered in a dosage of 1/10- 1/100of that of fasudil. In particular, it is preferred that the fasudilderivative is administered to the subject in a dosage of 10-1200 ng/kgbody weight per day, preferably in a dosage of 12.5-800 ng/kg bodyweight per day, such as in a dosage of 15-600 ng/kg body weight per day,more preferably in a dosage of 17.5-500 ng/kg body weight per day, evenmore preferably in a dosage of 20-400 ng/kg body weight per day, andmost preferably in a dosage of 24-360 ng/kg body weight per day. Theactual dosage regimen will be determined by the attending physician andclinical factors. As is well known in the medical arts, dosages for anyone patient depends upon many factors, including the patient's size,body surface area, age, the particular compound to be administered, sex,time and route of administration, general health, and other drugs beingadministered concurrently.

Further, it could be demonstrated that fasudil is capable of improvingthe motor coordination in a subject suffering from ALS, or in a subjectprone to suffer from ALS. In view of this finding, it is plausible thatthe same advantageous effect can be achieved with a pharmaceuticallyacceptable salt of fasudil, or with a fasudil derivative, e.g. withhydroxyl-fasudil or dimethyl-fasudil, or a pharmaceutically acceptablesalt of said derivative.

Accordingly, in a further aspect, the invention provides a method ofimproving motor coordination in a subject suffering or prone to sufferfrom sporadic or familial ALS, wherein the method comprisesadministering a pharmaceutical composition, comprising or consisting offasudil, or a fasudil derivative selected from hydroxy-fasudil anddimethyl-fasudil, or a pharmaceutically acceptable salt thereof, to thesubject.

The preferred embodiments according to this additional aspect areidentical with the preferred embodiments of the first aspect, and applylikewise. In particular, it is preferred that the ALS, such as thesporadic or familial ALS, is early stage ALS as diagnosable by theAwaji-shima diagnostic criteria, as further defined above. Further, theadministration of the pharmaceutical composition is startedpresymptomatically, and the subject is preferably a female. Although thepharmaceutical composition may be administered by various routes, it isparticularly preferred that the pharmaceutical composition isadministered orally. With regard to the dosage, if the pharmaceuticalcomposition comprises or consists of fasudil, it is preferred that thefasudil is administered to the subject in a dosage of 1-12 mg/kg bodyweight per day, preferably in a dosage of 1.25-8 mg/kg body weight perday, such as in a dosage of 1.5-6 mg/kg body weight per day, morepreferably in a dosage of 1.75-5 mg/kg body weight per day, even morepreferably in a dosage of 2-4 mg/kg body weight per day, and mostpreferably in a dosage of 2.4-3.6 mg/kg body weight per day.Alternatively, if the pharmaceutical composition comprises or consistsof a fasudil derivative, such as hydroxy-fasudil or dimethyl-fasudil,the fasudil derivative is administered to the subject in a dosage of10-1200 ng/kg body weight per day, preferably in a dosage of 12.5-800ng/kg body weight per day, such as in a dosage of 15-600 ng/kg bodyweight per day, more preferably in a dosage of 17.5-500 ng/kg bodyweight per day, even more preferably in a dosage of 20-400 ng/kg bodyweight per day, and most preferably in a dosage of 24-360 ng/kg bodyweight per day. Finally, it is further preferred that the fasudil,fasudil derivative, or pharmaceutically acceptable salt thereof isformulated as a formulation with sustained release or prolonged release,as further described above.

The improvement of the motor coordination in a subject may be determinedin accordance with a score as defined by the ALS functional ratingscale, as revised in accordance with Cedarbaum 1999 (Cedarbaum, Stambleret al. 1999):

-   1. Speech: Normal speech processes (4); Detectable speech    disturbance (3); Intelligible with repeating (2); Speech combined    with nonvocal communication (1); Loss of useful speech (0)-   2. Salivation: Normal (4); Slight but definite excess of saliva in    mouth, may have nighttime drooling (3); Moderately excessive saliva,    may have minimal drooling (2); Marked excess of saliva with some    drooling (1); Marked drooling, requires constant tissue or    handkerchief-   3. Swallowing: Normal eating habits (4); Early eating    problems—occasional choking (3); Dietary consistency changes (2);    Needs supplemental tube feeding (1); NPO—exclusively parenteral or    enteral feeding (0)-   4. Handwriting: Normal (4); Slow or sloppy—all words are legible    (3); Not all words are legible (2); Able to grip pen but unable to    write (1); Unable to grip pen (0)-   5. Cutting food and handling utensils:    -   a. Patients without gastrostomy: Normal (4); Somewhat slow and        clumsy, but no help needed (3); Can cut most foods, although        clumsy and slow, some help needed (2); Food must be cut by        someone, but still can feed slowly (1); Needs to be fed (0)    -   b. Patients with gastrostomy: Normal function (4); Clumsy but        able to perform all manipulations independently (3); Some help        needed with closures and fasteners (2); Provides minimal        assistance to caregiver (1); Unable to perform any aspect of        task (0)-   6. Dressing and hygiene: Normal function (4); Independent and    complete self-care with effort or decreased efficiency (3);    Intermittent assistance or substitute methods (2); Needs attendant    for self-care (1); Total dependence (0)-   7. Turning in bed and adjusting bed clothes: Normal (4); Somewhat    slow and clumsy, but no help needed (3); Can turn alone or adjust    sheets, but with great difficulty (2); Can initiate, but not turn or    adjust sheets alone (1); Helpless (0)-   8. Walking: Normal (4); Early ambulation difficulties (3); Walks    with assistance (2); Nonambulatory functional movement (1); No    purposeful leg movement (0)-   9. Climbing stairs: Normal (4); Slow (3); Mild unsteadiness or    fatigue (2); Needs assistance (1); Cannot do (0)-   10. Dyspnea: None (4); Occurs when walking (3); Occurs with one or    more of the following: eating, bathing, dressing (2); Occurs at    rest, difficulty breathing when either sitting or lying (1);    Significant difficulty, considering using mechanical respiratory    support (0)-   11. Orthopnea: None (4); Some difficulty sleeping at night due to    shortness of breath, does not routinely use more than two pillows    (3); Needs extra pillows in order to sleep (more than two) (2); Can    only sleep sitting up (1); Unable to sleep (0)-   12. Respiratory insufficiency: None (4); Intermittent use of BiPAP    (3); Continuous use of BiPAP during the night (2); Continuous use of    BiPAP during the night and day (1); Invasive mechanical ventilation    by intubation or tracheostomy (0)

In a final aspect, the present invention provides a method of prolonginglifetime of a subject suffering or prone to suffer from sporadic orfamilial ALS, wherein the method comprises administering apharmaceutical composition, comprising or consisting of fasudil, or afasudil derivative selected from hydroxy-fasudil and dimethyl-fasudil,or a pharmaceutically acceptable salt thereof, to the subject.

The preferred embodiments according to this additional aspect areidentical with the preferred embodiments of the first aspect, and applylikewise. In particular, it is preferred that the ALS, such as thesporadic or familial ALS, is early stage ALS as diagnosable by theAwaji-shima diagnostic criteria, as further defined above. Further, theadministration of the pharmaceutical composition is startedpresymptomatically, and the subject is preferably a female. Although thepharmaceutical composition may be administered by various routes, it isparticularly preferred that the pharmaceutical composition isadministered orally. With regard to the dosage, if the pharmaceuticalcomposition comprises or consists of fasudil, it is preferred that thefasudil is administered to the subject in a dosage of 1-12 mg/kg bodyweight per day, preferably in a dosage of 1.25-8 mg/kg body weight perday, such as in a dosage of 1.5-6 mg/kg body weight per day, morepreferably in a dosage of 1.75-5 mg/kg body weight per day, even morepreferably in a dosage of 2-4 mg/kg body weight per day, and mostpreferably in a dosage of 2.4-3.6 mg/kg body weight per day.Alternatively, if the pharmaceutical composition comprises or consistsof a fasudil derivative, such as hydroxy-fasudil or dimethyl-fasudil,the fasudil derivative is administered to the subject in a dosage of10-1200 ng/kg body weight per day, preferably in a dosage of 12.5-800ng/kg body weight per day, such as in a dosage of 15-600 ng/kg bodyweight per day, more preferably in a dosage of 17.5-500 ng/kg bodyweight per day, even more preferably in a dosage of 20-400 ng/kg bodyweight per day, and most preferably in a dosage of 24-360 ng/kg bodyweight per day. Finally, it is further preferred that the fasudil,fasudil derivative, or pharmaceutically acceptable salt thereof isformulated as a formulation with sustained release or prolonged release,as further described above.

The following examples are intended to further illustrate the invention.

DESCRIPTION OF THE FIGURES

FIG. 1: Kaplan-Meier time-to-failure plot for onset of symptomaticneurological disease in SOD1^(G93A) mice treated from d50 (A) or fromd80 (B) on differentiated by gender. VEH: solid line; Fas30: dashedline; Fas100: pointed line.

FIG. 2: Kaplan-Meier survival plot for age at death in SOD1^(G93A) mice.VEH: solid line; Fas30: dashed line; Fas100: pointed line.

FIG. 3: Kaplan-Meier survival plot for age at death in pre- andsymptomatically treated male and female SOD1^(G93A) mice. VEH: solidline; Fas30: dashed line; Fas100: pointed line.

FIG. 4: Disease duration for pre- and symptomatically treated male andfemale SOD 1^(G93A) mice. VEH: solid line; Fas30: dashed line; Fas100:pointed line.

FIG. 5: Kaplan-Meier time-to-failure plot for 180 s rotarod performancein pre- and symptomatically treated male and female SOD1^(G93A) mice.VEH: solid line; Fas30: dashed line; Fas100: pointed line.

FIG. 6: Kaplan-Meier time-to-failure plot for 180 s hanging wireperformance in pre- and symptomatically treated male and femaleSOD1^(G93A) mice. VEH: solid line; Fas30: dashed line; Fas100: pointedline.

FIG. 7: Average count of ChAT positive motoneurons per lumbar spinalcord section for presymptomatically treated male and female SOD1^(G93A)mice at day 100.

FIG. 8: Example of size measurement of ChAT positive motoneuron cellbody area per lumbar spinal cord section in female SOD1^(G93A) mice atday 100.

FIG. 9: Differentiation of ChAT positive motoneurons by size range infemale SOD1^(G93A) mice at day 100. From left to right each size range:wt; tg Veh; tg Fas30.

FIG. 10: Cumulative cell size per ventral horn section in femaleSOD1^(G93A) mice at day 100.

FIG. 11: (a): Average count of ChAT positive motoneurons per lumbarspinal cord section for presymptomatically treated male and femaleSOD1^(G93A) mice at time of death. (b): Average count of ChAT positivemotoneurons per lumbar spinal cord section for presymptomatically andsymptomatically treated male and female SOD1^(G93A) mice at time ofdeath. From left to right: Veh; Fas30; Fas100.

FIG. 12: Astrocytosis in SOD G93A transgenic mice treated with Vehiclecontrol (1A-3A), Fas30 (1B-3B) or Fas100 (1C-3C), immunohistochemical(IHC) labeling control without primary antibody (1D-3D). 1A-D: Spinalcord axial sections in phase contrast (overview). 2A-D Spinal cord axialsections labeled with GFAP/Cy2 (overview). The corresponding insets ofthe ventral horn are displayed in a higher magnification in 3A-D, dashedlines mark the border of the ventral horn. Scale bar in 2 and 3 is 200μm.

FIG. 13: GFAP positive astrocytes per 0.2 mm² in the anterior horn oflumbar spinal cord in wt and presymptomatically treated tg femaleSOD1^(G93A) mice at day 100.

FIG. 14: Numbers of GFAP positive astrocytes per 0.2 mm² of ventral hornof lumbar spinal cord in SOD1^(G93A) mice.

FIG. 15: Total axon numbers in the sciatic nerve of female SOD1^(G93A)mice at day 100.

FIG. 16: Axon numbers in the sciatic nerve of female SOD1^(G93A) mice atday 100 differentiated by size range. From left to right each sizerange: wt; tg Veh; tg Fas30.

FIG. 17: Survival of KA exposed motoneurons in astrocyte-motoneuronco-cultures of control and Fasudil treated conditions.

FIG. 18: Survival of KA exposed motoneurons in mono-culture of controland Fasudil treated conditions.

FIG. 19: Kaplan-Meier survival plot for age at death in male and femaleSOD1^(G93A) mice presymptomatically treated with Y-27632 (10 mg/kg bodyweight).

FIG. 20: Infiltration of Iba1 immunopositive microglial cells in thespinal cord anterior horn is modulated by ROCK inhibition. (A) Averagenumber of Iba1 immunopositive microglia per 0.5 μm² in the spinal cordanterior horn in SOD1^(G93A) mice treated with vehicle, Fas30 (Fasudil30 mg/kg/d) or Fas100 (Fasudil 100 mg/kg/d) at disease end-stage. (B)Average number of Iba1 immunopositive microglia per 0.5 μm² in thespinal cord anterior horn in vehicle treated wildtype mice and inSOD1^(G93A) mice treated with vehicle or Fas30 (Fasudil 30 mg/kg/d) atd100. (C,D) Representative images of immunofluorescent labelling of Iba1positive microglia in spinal cord anterior horn at end-stage (C) or atd100 (D) (Iba1/Cy3 (red) and DAPI (blue)). Bar represents means±SEM.n.s. not significant; * p<0.05; ** p<0.01; *** p<0.001. Scale bars, 100μm.

FIG. 21: Morphologic activatory changes of microglia in lumbar spinalcord after ROCK inhibition. (A) Quantitative analysis of relative cellnumbers belonging to three different morphologic subtypes (thinramified, stout ramified, rounded) of Iba1 immunopositive microglia inSOD1^(G93A) mice treated with vehicle, Fas30 (Fasudil 30 mg/kg/d) orFas100 (Fasudil 100 mg/kg/d) at end-stage. (B) Quantitative analysis ofmicroglia morphology as in (A) for vehicle treated wildtype mice andSOD1^(G93A) mice treated with vehicle or Fas30 (Fasudil 30 mg/kg/d) atd100. (C) Exemplary high magnification microscopic images of microgliamorphologic types analyzed (Iba1/Cy3 (red) and DAPI (blue)). Barrepresents means±SEM. n.s. not significant; * p<0.05; *** p<0.001. Scalebar, 50 μm.

FIG. 22: Reduction of microglial cytokine/chemokine release by combinedLPS+Fasudil treatment.

EXAMPLES Example 1

Material and Methods

Animals

All animal experiments were carried out according to the regulations ofthe local animal research council and legislation of the State of LowerSaxony. High-copy B6SJL-Tg(SOD1*G93A)1Gur/J were obtained from JacksonLabs (Stock Number 002726; Bar Harbor, USA) that had originally beenproduced by Gurney et al. (Gurney, Pu et al. 1994). The colony wasmaintained by crossing C57BL/6 males harboring the transgene withwild-type SJL females. To verify presence of the transgene in the F1progeny, tail biopsies were collected from 14-day-old pups and genotypedusing PCR. At the age of 35-45 days mice were moved to the testingfacility to acclimate to the new environment with a 12-h light/darkcycle before being assigned to a study.

Experimental Groups

At 50 days (presymptomatic, d50) or 80 days of age (symptomatic, d80)mice were separated into Fasudil-30 (Fas30)- and Fasudil-100(Fas100)-Treatment or Vehicle Control (Veh) groups. Prior to anytreatment, groups were constituted so as to minimize between-groupvariability by using the following criteria. Groups were balanced withrespect to gender and body weight within gender. In addition, groupswere age-matched and littermate-matched. Each male and female in theTreatment group had a corresponding male and female littermate in theVehicle Control group. The study was observer-blinded.

Treatments

Before treatment initiation the inventors monitored the drinking amountsof mice that were determined with 0.2 ml/g body weight/day. To reachsufficient CSF levels of Fasudil the inventors administered Fasudil (LCLabs, Woburn, USA; product number F-4660) at a low concentration of 15mg (Fas30) or at a high concentration of 50 mg (Fas100) in 100 mldrinking water. Control groups received the respective amount ofisotonic drinking water termed as vehicle (Veh). CSF analysis aftersub-occipital cistern magna puncture demonstrated that Fasudil readilycrossed the blood-brain-barrier (data not shown).

Chronic oral treatment with a low dose of Fasudil (F30) or a high doseof Fasudil (F100) was started either presymptomatically at day 50 orsymptomatically at day 80 and continued until death.

Monitoring Disease Progression

Neurological Score. Neurological scores were assessed every three daysfor each mouse from 50 days of age. The neurological score employed ascale of 0 to 4 that had been developed in ALS mouse trials before.Score criteria used to assign each score level were:

-   4 Full extension of hind legs away from lateral midline when mouse    is suspended by its tail, and mouse can hold this for 2 seconds,    suspended 2-3 times.-   3 Collapse or partial collapse of leg extension towards lateral    midline (weakness) or trembling of hind legs during tail suspension.-   2 Toes curl under at least twice during walking of 10 cm, or any    part of foot is dragging along cage bottom/table.-   1 Rigid paralysis or minimal joint movement, foot not being used for    forward motion*. * If score criterion 2 is reached, food pellets are    left on bedding and water is additionally placed in a well on the    bedding.-   0 Mouse cannot right itself within 30 seconds from either side.

Body Weight. Body weight is a sensitive indicator of any malaise thatmight result from chronic drug treatment and of motor impairment thatoccurs during disease progression. Body weight measurements wererecorded every three days for each animal beginning at 50 days of age.

Survival. Date and cause of death were recorded for each mouse. Forhumane reasons, animals are closely monitored and sacrificed as moribundprior to actual death using criteria for severe moribundity. Todetermine duration of survival reliably and humanely, the moribundstate, defined as the inability of mice to right themselves 30 secondsafter being placed on a side (a neurological score of 4) was used. Themoribund mice were scored as “dead”, and were euthanized using carbondioxide.

Disease duration. This parameter was selected in order analyze thetreatment effects under a clinically relevant situation of the time fromdisease onset until death.

Immunohistochemistry of Spinal Cord, Sciatic Nerve and GastrocnemiusMuscle Sections

After deep anaesthesia, mice were transcardially perfused with PBSsolution followed by 4% paraformaldehyde. Spinal cords, sciatic nervesand gastrocnemius muscles were removed. Spinal cords and gastrocnemiusmuscles were then postfixed for 2 h in 4% paraformaldehyde. Thereafter,the tissue was dehydrated in 30% sucrose overnight and kept at −20° C.until further processing. Coronal sections of the lumbar spinal cord(L3-L6) (20 μm) and horizontal sections of the tibial muscle (35 μm)were prepared using a Leica Kryostat and collected on gelatine-coatedglass slides.

Spinal cord sections selected for immunohistochemical processing werefirst rehydrated. Then antigen-retrieval was performed for 4 h in TBS at60° C. for later GFAP or Iba1 labeling and for 30 min in sodium citrate(pH 9.0) at 80° C. for later ChAT labeling. After washing for one timein PBS the sections were incubated at room temperature either in PBS and10% of normal goat serum for later GFAP labeling or in PBS and 5% normaldonkey serum for later ChAT labeling to block unspecific binding. Theprimary antibodies (anti-ChAT 1:100, AB144 P, Millipore; anti-GFAP1:200, DAKO; anti-Iba1 1:500, WAKO, Osaka, Japan) were applied at 4° C.for one night for GFAP and for three nights for ChAT. The secondaryantibodies (Cy3 anti-goat or Cy2/Dylight anti rabbit; both Dianova) wereapplied on separate sections 1:300 for 1 h at room temperature. Then anuclear counter-stain with DAPI (4,6-diamidino-2-phenylindole)(Sigma-Aldrich) was performed before mounting in Moviol (Hoechst,Frankfurt, Germany).

Sciatic nerves were removed separately and postfixed for 24 h in 4%paraformaldehyde. For embedding, the samples were fixed in 3%Glutaraldehyd in PBS for 3 h at 4° C., then postfixed in 1% Osmiumoxidin PBS for 1 h at 4° C. After three washing steps the nerves wereembedded in Araldit and Propylenoxide with a ratio of first 1:1 and then2:1, each for 35 min. Before putting into a flat mould for drying andharding, Propylenoxide had to evaporate under a hood for 1 h. Thesciatic nerves embedded in araldit blocks were cut with a Microtom(Leica Ultracut) into semithin sections (380 nm) and were collected ongelatine coated glass slides. Then they were stained withRichardson-Solution (2:1:1 of 1% Azur-2-solution, 2% Methylbluesolutionand 1% Borax-solution (Di-Natriumtetraborat-10-hydrat)). After washingin distilled water, slices were dried and mounted in Depex.

Gastrocnemius muscle sections were rehydrated and permeabilization wasdone for 2 h at room temperature in PBS, 5% IgG free and protease freeBovine Serum Albumin (Jackson ImmunoResearch Laboratories, Lot 91563),0.5% Trition X-100 (Molecular Sigma biology, T-8787). The primaryantibodies (rabbit anti-Neurofilament M, 1:1000, Millipore, AB1987;rabbit anti-VAChT, 1:1000, Sigma-Aldrich, V5387) were applied togetherat 4° C. overnight in 1% BSA, 0.25% Triton X-100. After extensivewashing in PBS 1% BSA, the secondary antibody (anti-rabbit Alexa fluo488, 1:300; A11034, Invitrogen Molecular Probes) was incubated withα-bungarotoxin Alexa Fluor 594 conjugate (1:1000, B13423, InvitrogenMolecular Probes) for 90 min in PBS, 1% BSA at room temperature. Finallythe muscle sections were washed for three times in PBS, once in waterand were then mounted in Moviol (Hoechst, Frankfurt, Germany).

Quantitative Evaluation of Spinal Cord Motoneurons, Astro- andMicroglia, Sciatic Nerve Axons and NMJ

In order to quantify motoneuron numbers in coronal cryosections from thelumbar spinal (L3-L6) cord first the area of the ventral horn wasdefined according to the criteria from Kong et al. (Kong and Xu 1998).Cells were counted as motoneurons if they were located in the ventralhorn, had a clearly identifiable nucleolus, were at least 200 μm² insize and were ChAT-positive (Pitzer, Kruger et al. 2008). At least fivesections per mouse spinal cord that were 100 μm apart over a length ofat least 500 μm isolated from the lumbar spinal cord were counted. Inthe d50 treatment group analyzed at death, a total of 20 mice wascounted from the vehicle (3 male, 3 female), Fas30 (3 male, 3 female),Fas100 (3 male, 3 female) and wildtype (1 male, 1 female) group. In thed80 treatment group analyzed at death, a total of 20 mice was countedfrom the vehicle (3 male, 3 female), Fas30 (3 male, 3 female), andFas100 (3 male, 3 female) and wildtype (1 male, 1 female) group. In thed50 treatment group analyzed at d100, a total of 20 mice was countedfrom the vehicle (3 male, 3 female), Fas30 (3 male, 3 female), Fas100 (3male, 3 female) and wildtype (1 male, 1 female) group.

Astrogliosis and microgliosis were evaluated by the quantification ofGFAP or Iba1 positive cells in the ventral horn of lumbar spinal cordsections, respectively. Again, the area of the ventral horn was definedand GFAP or Iba1 positive cells were manually counted. In the d50treatment group analyzed at death, a total of 20 mice was counted fromthe vehicle (3 male, 3 female), Fas30 (3 male, 3 female), Fas100 (3male, 3 female) and wildtype (1 male, 1 female) group. In the d50treatment group analyzed at d100, a total of 20 mice was counted fromthe vehicle (3 male, 3 female), Fas30 (3 male, 3 female), Fas100 (3male, 3 female) and wildtype (1 male, 1 female) group.

For the evaluation of microglia morphology at least 10 randomly chosencells per ventral horn were examined with a 63× objective. Cells wereclassified as “thin ramified”, “stouter ramified” and “rounded”according to a recent morphologic description by Graeber et al.(Graeber, M. B. (2010) Science 330(6005): 783-788). A ramified cell wasdefined as a cell that has at least 2 processes that are at least twicethe size of cell body. Thin ramified cells were defined to have a smalldiameter of ramifications (<0.5 μm) and a small cell soma diameter (<5μm). Stoutly ramified cells were defined with thicker ramifications(>0.5 μm) and a larger cell soma diameter (>5 μm). Round cells weredefined to exhibit no processes and a round appearance.

Semithin cuts of sciatic nerve axons were evaluated from lightmicroscopy images for axonal numbers and axoplasm size with thesemi-automated counting and size measuring tool of the Image J software.In the d50 treatment group analyzed at d100, a total of 17 female micewere counted from vehicle (5), Fas30 (7), and wildtype (5).

In order to evaluate the integrity of NMJ, muscular sections, that hadbeen labeled for the distal axonal and endplate innervating structureswith anti-Neurofilament M/Alexa 488 and anti-VAChT/Alexa 488 and forendplates with α-bungarotoxin/rhodamine, respectively, were examinedunder a fluorescence microscope. End-plates were scored as “innervated”if there was complete overlap with the axon terminal, or “denervated” ifthe end-plate was not associated with an axon. Some neuromuscularjunctions that presented a preterminal axon only, or showed only apartial overlap between end-plate and terminal were labeled as“intermediate”. For each muscle 4 sections of its distal, middle andproximal part that were 140 μm apart over a length of at least 560 μmand contained at least 10 endplates per section were evaluated.

Electrophysiology

Nerve conduction velocities (NCVs) and compound muscle action potentials(CMAPs) were determined at 100 days of age as previously described(Meyer zu Horste, Prukop et al. 2007). Briefly, mice were anaesthetizedusing an intraperitoneal injection of ketamin (100 mg/kg) and xylazin (5mg/kg), while body temperature was maintained constantly using a heatingplate connected to a rectal temperature sensor (cma, Stockholm, Sweden).Tail NCV proved to be more reproducible than recordings from the sciaticnerve which had to be exposed surgically. Stimulation was performed withincreasing voltage until supramaximal stimulation was achieved. MaximumCMAP voltage was recorded from the tail muscle with fine subcutaneousneedle electrodes using a Jaeger-Toennies Neuroscreen instrument(Würzburg, Germany). CMAP amplitudes were calculated as peak to peak.NCV was calculated from distance and motor latency differences betweenproximal and distal stimulations.

Statistical Analysis

The data were then analyzed using Kaplan-Meier survival fit analysiswith the Log-Rank and Wilcoxon tests for statistical significance. Coxproportional hazards analysis was also performed to determine hazardratios and test for statistical significance of their differences usingthe Effect Likelihood Chi Square test. Statistical analyses wereperformed using Statistica 9.1 (StatSoft, Hamburg, Germany). P-valuesless than 0.05 were taken to be statistically significant.

Results

Disease Onset is not Modified by Pre- or Symptomatic Fasudil Treatment

Onset of disease is defined as reaching neurological score 3 (collapseor partial collapse of leg extension towards lateral midline (weakness)or trembling of hind legs during tail suspension). There was nostatistically significant difference in the onset of disease in animalstreated from d50 or from d80 differentiated by gender (FIG. 1).

Survival and Disease Duration are Prolonged in Presymtomatically TreatedSOD G93A Mice

The proportion of mice surviving over time non-differentiated by genderis shown in FIG. 2. Whereas both symptomatic (d80) Fas30 and Fas100groups did not show a statistically significant difference in survivalproportions over time, the presymptomatic (d50) Fas30 group profitedsignificantly from the treatment.

If differentiated by gender, the female presymptomatic Fas30 group wassignificantly profiting from treatment surviving in average for 141.8days compared to 130.4 days in the control group. Presymptomatic Fas30treatment for males showed a trend in prolongation of survival.

If Fasudil treatment was initiated only from a symptomatic time pointon, it was not able to prolong survival in either female or male group.

Disease duration is defined as beginning from the appearance of thefirst clinical symptoms until death. In this parameter thepresymptomatically treated female mice showed a prolonged time. Thesymptomatically treated groups did not show any significant alterations.

The clinical monitoring of the therapy study consisted of clinicalneurologic scoring and body weight analysis performed every three daysfor all animals. Thereby, the inventors were able to exactly define theonset of disease, monitor the disease progression and determine the timeof death. In presymptomatically treated animals the inventors did notobserve a prolongation of disease onset. This is a common finding inmany other ALS therapy studies, e.g. using Riluzole.

The criteria to terminate the therapy study (later referred to as timeof death) were fulfilled if the animals had reached the terminalneurological scoring or had lost a defined percentage of the maximalbody weight. This time point then reflects the survival time of theanimals whereas disease duration was measured from onset of diseaseuntil time of death. The presymptomatic treatment group significantlyprofited from the Fas30 treatment concerning survival, whereas theeffect of Fas100 was less prominent. If differentiated for gender, thissurvival promoting effect was most pronounced for females. Without beingbound by theory, an explanation for this can be inferred already fromother studies in which the inventors have tested pharmacologic ROCKinhibitors in various experimental settings before. Here, the inventorshad noted an important dose-dependent effect of ROCK inhibition byFasudil (HA-1077) and more importantly by its derivativeDimethyl-Fasudil (HA-1152) (Lingor 2007, 2008). It seemed that for thesepharmacologic ROCK inhibitors an optimal concentration has always to betitrated depending on the experimental application because thetherapeutic window of ROCK inhibitors seems to be limited. As anexample, the regenerative potential of ROCK inhibition to increase thenumber of regenerating axons after optic nerve crush lesion wassignificantly reduced if a high intravitreal dose of Dimethylfasudil (40μmol/l) had been applied in comparison to a medium dose (400 nmol/l)(Lingor, Teusch et al. 2007). The inventors attributed this decrease inoutgrowth promoting activity to a co-inhibitory activity ongrowth-related kinases, such as protein kinase N, protein kinase A,mitogen- and stress-activated protein kinase 1, citron kinase or myosinlight chain kinase (Davies, Reddy et al. 2000).

Interestingly, presymptomatic ROCK inhibition with the pharmacologicallymore potent ROCK inhibitor Y-27632 in a dosage comparable in efficacywith Fasudil low dose did not result in survival promoting effects (FIG.19). Thus, the beneficial effects of Fasudil on survival do not seem tobe a group effect of ROCK inhibitors but is likely to be specific forFasudil.

The symptomatic (d80) treatment was not able to improve survival in bothapplied Fasudil concentrations. This could be due to an already too farprogressed disease process that Fasudil is not able to halt. Anotherpossibility would be that Fasudil is not as effective against end-stagedisease pathophysiology as against the early stage alterations. Whereasearly stages are characterized by destruction of NMJ innvervation andaxonal degeneration, progression of symptomatic disease until end-stageis mainly characterized by infiltration of microglia and overallinflammation. In comparison, many other treatments that have beensuccessful if started presymptomatically have failed if applied only atsymptomatic stages. Therefore, a very early diagnosis is most relevantin this disease so that Fasudil is able to exert its beneficial effectsover a longer time period.

Motor Coordination but not Overall Muscular Strength is Improved byPresymptomatic Fasudil Treatment

From a patient's point of view overall survival is not the onlyparameter of a successful therapy. Many recent therapeutic studies inhumans rather evaluate an increased time with improved quality of life.An improved motor function could be one parameter that is relevant forthe quality of daily life and therefore the inventors chose twodifferent tests to monitor motor function in the present animal model.The rotarod test evaluates fine motor coordination and is a verysensitive test for the ability to move on an unstable underground.

Evaluating motor coordination by subjecting the animals to the rotarodtest, the presymptomatic female treatment group failed later in the taskto reach a total running time of 180 s (122.5 days) in comparison to thevehicle-treated group (114.2 days). For males, there was only a slightnon-significant trend to improved performance in the presymptomaticFas30 Group (FIG. 5). Wildtype animals always reached the maximum timeof 180 s (data not shown).

Presymptomatically treated mice showed a prolonged time to the firstfailure in the rotarod test. This effect was most pronounced for femalemice and moderately improved in male mice. A symptomatic treatment wasnot able to improve rotarod performances.

The hanging wire test evaluates crude grip strength of the upperextremities. It does not allow differentiating between only minordeficits in strength because the body weight of the animals is inrelation to the grip strength relatively heavy.

The hanging wire performance evaluating crude muscular strength in thetask to reach a total hanging wire time of 180 s was not different ineither treatment groups of both sexes (FIG. 6). Wildtype animals alwaysreached the maximum time of 180 s (data not shown).

In both presymptomatic and symptomatic treatment groups the time tofailure in this test was prolonged only to a very moderate but notsignificant amount in comparison to controls. Thus Fasudil seems torather improve fine motor coordination than crude muscular strength.

Motoneurons of Female SOD G93A Mice are Protected by PresymptomaticFasudil Treatment

In order to correlate the clinical and behavioural results to theneurodegenerative disease process on a histological level the inventorschose to evaluate first the survival of spinal cord motoneurons andsecond of spinal cord astrogliosis. The analysis of ChAT positivemotoneurons in the spinal cord represents a basic marker of ALSpathology, however, does not necessarily reflect the functionalintegrity of this principal part of the motor unit. Whereas theinventors had undertaken the clinical and behavioural testingsrepeatedly during the entire disease process the inventors were confinedto one time point of each experimental group to perform theimmunohistochemical analysis after sacrification of the animal. Asclinical disease is already very obvious at d100 the inventors chosethis time point for analysis and additionally examined all animals attime of death.

Motor function is dependent on the number of functional motor units thatare comprised of the motoneuron, its axon, the neuromuscular junctionand the innervated muscle fibers. As the number of motoneuron cellbodies is a critical component of these motor units and ROCK inhibitionhas shown to be able to confer a cytoprotective effect on neuronal cellsthe inventors compared the numbers of ChAT positive motoneurons betweentreatment groups. Therefore, the inventors analyzed motoneuron numbersof treated animals at day 100 of life and at time of death which canhowever be different for the individual animal (compare FIG. 3).

The motoneuron analysis performed at day 100 for presymptomaticallytreated female mice showed only a minor loss of motoneuron numbersbetween wildtype and transgenic vehicle treated mice. Obviously, theloss of motoneurons is only moderate at that early time point ofclinical disease. If transgenic animals were compared that receivedeither vehicle or Fas30, the latter ones showed a strong trend ofelevated numbers of motoneurons (FIG. 7).

Because motoneurons exhibit large motoneuron cell bodies and it isgenerally acknowledged that the size of the cell body can also be seenas a marker for intact cell viability, the inventors measured the sizesof all motoneurons in the different animal groups (example in FIG. 8).

If the absolute numbers of motoneurons were differentiated by cell bodysize transgenic animals had lesser motoneurons in all size groups.Although the transgenic Fas30 group displayed more motoneurons in allsize groups, this trend was not significant (FIG. 9).

If cumulative values of the cell body size per anterior horn sectionwere compared between groups, the cumulative size in the transgenicFasudil group showed a defined trend to be larger than the vehicletreated group (FIG. 10).

Whereas motoneuron cell numbers were not significantly altered in bothtransgenic treatment groups at day 100, the presymptomatically treatedanimals analyzed at time of death showed significant differences. Incontrast to the low number of ChAT cell numbers per lumbar spinal cordsection in vehicle treated female mice (6.1±0.95), female Fas30 treatedanimals had a significantly increased motoneuron count (8.6±0.36) thatwas not found in the high dose F100 group (7.3±1.00). The presymptomaticmale groups did not profit from either Fas30 or Fas100 treatment (FIG.11a ). However, the symptomatic male treatment group exhibited highermotoneuron numbers with Fas30 treatment than the vehicle group. Thefemale symptomatic treatment groups did not differ regarding motoneuroncell counts (overview of all data in FIG. 11b ).

Presymptomatically Fasudil treated mice showed a trend of increasednumbers of spinal cord motoneurons at day 100, however, that was notsignificant as also in comparison to wildtype animals most motoneuronswere not yet severely affected by the disease process. The analysis attime of death resulted in increased motoneurons numbers for female mice.The symptomatic treatment was not able to improve motoneuron survival.

Astrocytosis is Reduced in Presymptomatically Treated Fasudil Mice

Reactive astrogliosis represents another parameter in the evaluation ofhistologic manifestation of ALS disease. In human ALS patients, theclinical progression of this neurodegenerative disease seems to beassociated with changes in astrocytic cell infiltration in the spinalcord. Therefore, the inventors evaluated the extent of astrogliosis inthe lumbar spinal cord by counting the number of GFAP positiveastroglial cells in the ventral horn and normalized values to area (FIG.12).

Comparable to the analysis of surviving motoneurons the inventorsevaluated animals at day 100 of life and at time of death. Astroglialcell numbers at day 100 in female mice between wildtype and transgenicanimals were significantly higher in the transgenic vehicle group. Incomparison to the transgenic vehicle treated animals Fas30 significantlyreduced the GFAP cell number (FIG. 13).

The quantitative analysis of presymptomatically treated mice at timepoint of death resulted in lower numbers of astrocytes in vehicletreated transgenic animals compared to day 100. Comparing the treatmentgroups of transgenic animals, numbers of GFAP positive astroglia wereslightly higher for the Fas30 male (38.2±4.4) but not for the Fas30female (31.6±5.9) group. The Fas100 groups (male 29.24±4.5; female27.9±4.0) did not differ from vehicle controls (male 31.3±4.7; female28.91±4.7) (FIG. 14).

By evaluating numbers of GFAP positive astroglia in the spinal cord thepresymptomatically treated female animals had significantly lessinfiltrating astroglia reflecting that this reactive astrogliosis hadnot as far evolved as in the untreated mice. The ability of Fasudil toinfluence inflammatory processes has also been demonstrated by othergroups. The analysis of presymptomatically treated mice at time of deathresulted in an unexpected finding. Here, both male and female miceshowed higher numbers of astroglia. The inventors attribute this findingto the prolonged life time of the animals and speculate that whereas atthe same time points like day 100 animals exhibit less astrogliosis withFasudil treatment they have a strong “extra” accumulation until endstage/time of death that is on average more than one week later than invehicle treated animals. It seems like a “bursting of a dam” takes placeand leads to an exponential astroglia infiltration in this very lateclinical stage. Interestingly, the prolonged time of life did notnegatively influence motoneuron numbers that rather seem to be morestably conserved from early time points on.

Moreover, motor function is dependent on the number of functional motorunits that are comprised not only of the spinal cord motoneuron itself,but also of its axon, the neuromuscular junction and the innervatedmuscle fibers. Therefore, the inventors did not only investigate spinalcord ALS pathology but also the remaining distal compartments of the PNSand innervated muscle.

However, evaluating sciatic nerve axonal pathology and NMJ integrity ofgastrocnemius muscle, there were no significant differences betweenFasudil and control treated animals.

In a corresponding in vitro paradigm of motoneuron survival, Fasudilrobustly protected motoneurons from cell death in a co-culture withastrocytes but also in mono-culture.

Axonal Degeneration is not Altered after Fasudil Treatment

The degeneration of motor axons is an early event in the development ofALS disease that has important functional consequences. Therefore, theinventors analyzed the extent of axonal degeneration in the sciaticnerve in wildtype and ALS mice at day 100 in females. The manualcounting of the total number of preserved axons in the sciatic nerveresulted in a significantly decreased axonal number in transgenicanimals in comparison to wildtype animals. The transgenic vehicle groupand the Fas30 group showed comparable axon numbers (FIG. 15).

If differentiated by axoplasmal diameter size range, the axonal numberin each size range was higher in wildtype than in transgenic animalsexcept for the size range smaller than 3.5 μm. There were no significantdifferences between transgenic vehicle treated and Fas30 treated animals(FIG. 16).

Comparative Example

If SOD G93A mice were treated with the pharmacologically more potentROCK inhibitor Y-27632 (10 mg/kg body weight) in a presymptomatictreatment paradigm from d50 on, there were no significant effects seenconcerning survival (cf. FIG. 19).

Example 2

Fasudil Protects Cultured Motoneurons from Kainic Acid Toxicity

In order to compare the Fasudil neuroprotective effect on motoneurons invivo with an corresponding in vitro paradigm the inventors subjectedwildtype primary motoneurons cultured in a astrocyte/motoneuronco-culture to kainic acid (KA) toxicity. KA exposure for 24 hdramatically decreased the number of viable motoneurons. However, ifcultures had been treated with Fasudil at 4 or 20 μM concentration, thereduction in viable motoneurons declined significantly less. ThusFasudil protected motoneurons from KA induced cell death (FIG. 17).

Fasudil was also effectively protecting mono-cultures of primarymotoneurons thus indicating that the protective effect does not dependon co-cultured astrocytes (FIG. 18).

In summary, Fasudil protected motoneurons from cell death in aco-culture with astrocytes but also in mono-culture in a correspondingin vitro paradigm of motoneuron survival.

Example 3

Materials and Methods

Microglial Cell Preparation, Culturing and Treatment

Primary cell cultures were prepared from whole brains of newborn (PO)mice and cultured in complete Dulbecco's modified Eagle's medium (DMEM,Invitrogen/Gibco, Karlsruhe, Germany), supplemented with 10% fetal calfserum (FCS, Invitrogen/Gibco), 100 U/ml penicillin and 100 μg/mlstreptomycin (both Biochrom, Berlin, Germany) as previously described(Regen, et al. (2011) Brain, Behavior, and Immunity 25(5): 957-970). Inbrief, brains of newborn B6/SJL SOD1G93A and from B6/SJL SOD1wt,respectively, were liberated from meninges and blood vessels, washedwith Hank's balanced salt solution (HBSS; Biochrom) and incubated with2.5% trypsin (Biochrom) for 10 min at 37° C. The enzymatic reaction wasstopped by addition of complete medium, supplemented with DNAse [5mg/ml] (Worthington Biochemicals, Lakewood, N.J., USA). Remaining cellclusters were mechanically separated and the suspension was centrifugedat 900 rpm at 4° C. for 10 min. The supernatant was removed, cells wereresuspended in fresh complete medium and seeded in 75 cm2, poly-L-lysine(PLL)-coated culture flasks. Immediately before being used, flasks wereincubated with 100 μg/ml PLL (Invitrogen/Gibco) for 30 min at RT andexcess of PLL was washed off by three rinses with sterile ddH2O.Subsequently, cultures were maintained in a humidified atmosphere with5% CO₂ at 37° C. On the next day, primary mixed-glial cultures werewashed three times (PBS, Invitrogen/Gibco) and received fresh completemedium, the latter being repeated the following day. One week afterseeding, microglial proliferation was stimulated by adding completemedium supplemented with 30% of L929-conditioned cell culturesupernatant (see section “L929 Mouse Fibroblast Cultures” below). Afteranother two days, microglial cells were harvested by shaking them offthe astrocytic layer, taking advantage of their different adhesionproperties. After washing with complete medium and counting, cells wereplated in 96-well plates at a density of 15,000 cells/well for ELISA orwere plated in 24-well plates at a density of 50,000 cells/well onPLL-coated coverslips for later immunocytochemical processing. Thepurity of the microglial preparations was routinely >98%, based onnuclear staining with DAPI, immunocytochemistry for CD11b and Iba1 aswell as Griffonia simplicifolia isolectin B4-based labeling (data notshown).

Primary cultures underwent five different general treatments: (a) Cellswere left untreated in medium serving as controls. (b) Cells werestimulated with the TLR4 ligand LPS (E. coli R515; Axxora/Appotech TLRligand set 1, APO.54N-018) for 18 hours at a final concentration of 10ng/ml. (c) Cells underwent a treatment with Fasudil at 2 μM, 10 μM or 50μM, respectively, for 18 hours. (d) Cells were stimulated with LPS, asdescribed above, in the presence of various concentrations of Fasudil.(e) Cells were preincubated for 1 h, 4 h or 12 h, respectively withFasudil at 50 μM, prior to a LPS challenge in the continued presence ofFasudil at 50 μM. Stimulation experiments were routinely carried outtwice.

Determination of Microglial Cell Vitality

Microglial viability was assayed as metabolic activity using WST-1reagent(4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate), based on the enzymatic cleavage of WST tetrazolium salt toformazan by the succinate-tetrazolium reductase system of therespiratory chain of intact mitochondria. The assay was performedaccording to the instructions of the manufacturer (Roche AppliedScience, Mannheim, Germany). After 3 h of incubation, the resultingcolor reaction was measured at 450 nm (with 655 nm as the referencewavelength) in a microplate reader (Bio-Rad, Munich, Germany).Accordingly, Fasudil was set to a maximum concentration of 50 μM, sinceup to this concentration, there was no reduction in metabolic activityobserved.

L929 Mouse Fibroblast Cultures

L929 fibroblasts were routinely cultured in complete medium and passaged(1:5) every two weeks. After 14 days of continuing cultivation (nomedium change in between), supernatants were isolated and stored at −20°C. until used for the stimulation of microglial proliferation. After 30passages, fresh L929 cultures were established from a stock stored inliquid nitrogen.

Cyto- and Chemokine Measurements (ELISA)

Following microglial stimulations and treatments, culture supernatantswere collected and stored at −20° C. until assayed. Supernatants wereanalyzed for the release of cyto- and chemokines by commercialenzyme-linked immunosorbent assay (ELISA) test systems. Levels of IL-6,CCL2 (monocyte chemoattractant protein, MCP-1), CCL3 (macrophageinflammatory protein, MIP-1α) and CCL5 (regulated upon activation normalT-cell expressed and presumably secreted, RANTES) and CXCL1 (Chemokine(C-X-C motif) ligand 1) were determined using DuoSet ELISA DevelopmentKits (R&D Systems). TNFα (tumor necrosis factor α) was determined usingMAXTM ELISA kit (Biolegend). Absorbance was measured at 450 nm (with a540 nm reference wavelength) using a microplate reader (Bio-Rad).

Results

Protective Properties of Fasudil May be Due to Activated Microglia ofthe Neuroprotective M2 Phenotype

Microglial activation is a neuropathological hallmark of ALS. Microgliacan have very distinct and different phenotypic states, and, dependingon the physiologic conditions, may exert either a toxic or protectiveeffect on neurons. Microglia of the M1 phenotype (classically activatedmicroglia) are cytotoxic due to the secretion of prinflammatorycytokines and reactive oxygen species. Microglia of the M2 phenotype(alternatively activated microglia) block proinflammatory responses andproduce increased levels of neurotrophic factors and anti-inflammatorycytokines (for a review, see Henkel et al. 2009). Up to now,manipulating these microglial neuroprotective or neurotoxic effectorfunctions and attributes remain elusive and poor understood.

The extent of microglial cell dissemination in the lumbar spinal cordwas evaluated with a marker of activated microglia (Iba1) as describedabove under the heading “Quantitative evaluation of spinal cordmotoneurons and astroglia, sciatic nerve axons and NMJ”.

The results are shown in FIG. 20. Whereas there were no grossdifferences in the amount of microglial cells in all treatment groups atdisease end-stage (vehicle group 43.2±4.2 cells/0.5 mm²; Fas30 group48.8±3.3 cells/0.5 mm²; Fas100 group 50.2±4.7 cells/0.5 mm²) (FIG. 20A,C), the intermediate analysis during the disease course at d100 showeda significantly higher number of Iba1 immunopositive microglial cells inFasudil 30 treated SOD1^(G93A) mice (39.5±3.9 cells/0.5 mm²) incomparison to vehicle treated SOD1^(G93A) mice (27.8±4.7 cells/0.5 mm²).Vehicle treated wildtype mice exhibited only very few spinal cordmicroglia (14.0±2.8 cells/0.5 mm²) (FIG. 20 B,D).

The increased appearance of microglial cells in the Fasudil 30 treatmentgroup at d100 could indicate a beneficial and neuroprotective M2microglial response (cf. Henkel et al. 2009).

In order to further characterize the differential microglial behaviourin the treatment groups at d100, we performed a morphologicsubclassification of microglial cells, as described above under theheading “Quantitative evaluation of spinal cord motoneurons andastroglia, sciatic nerve axons and NMJ”. It is known that microglialmorphology can be altered due to its activation state and cells mayexhibit shapes with predominantly thin ramified processes, withpredominantly stout ramified processes or with a round/amoeboid shape.The latter two morphologic cell types are considered to reflect a stateof enhanced microglial activation in comparison to a surveillance typewhich is characterized by smaller cell bodies and thin ramifiedprocesses (exemplary microscopic images are shown in FIG. 21 C).

At disease end-stage there were no gross alterations of microglialmorphology in all treatment groups. At this time point, themorphological type with stout ramified processes was mostly found (FIG.21 A). However, at d100 microglia of Fasudil 30 treated SOD1^(G93A) miceexhibited predominantly thin stout ramified processes (70.8±5.3%), lesscells with thin ramifications (28.7±5.3%) and very few rounded cells(0.5±0.1%). In contrast, vehicle treated SOD1^(G93A) mice had more cellswith thin (52.6±6.3%) than with stout ramifications (46.7±6.3%) and veryfew rounded cells (0.7±0.2%). The vehicle treated wildtype groupexhibited mostly thin ramified processes (95.0±0.8%), very few cellswith stout ramifications (5.0±0.8%) and no rounded cells (0.0±0.0%)(FIG. 21 B).

Thus, it seems that an increased activation of microglial cells takesplace, possibly exhibiting the neuroprotective M2 phenotype.

Primary cultures of newborn SOD1 wild-type control (SOD1 wt) orrespective mutant (from advanced clinical SOD1G93A mice) microglia(15,000 cells/well) were incubated in the simultaneous presence of LPS(10 ng/ml) and different Fasudil concentrations (2 μM, 10 μM, 50 μM) for18 hours, respectively. Cytokine/chemokine release profile wasdetermined in the supernatants. The results are shown in FIG. 22. Dataare presented relative to the corresponding value of LPS-triggeredcytokine/chemokine release within each genotype, respectively (set as100%). Data are mean±SEM of quadruplicates of 2 independent experiments.

Whereas Fasudil at concentrations of 2 μM and 10 μM was not efficient insignificantly reducing the harmful proinflammatory cytokine release (cf.Henkel et al. 2009) of both, SOD1 wt and SOD1G93A microglia, the dose of50 μM Fasudil markedly reduced the release of TNFα, IL-6, CCL3, RANTESand CXCL1 in both genotypes. These factors are generally supposed inmediating an harmful inflammatory response. In contrast, values for CCL2were slightly increased (+8.9%)/unaffected at this dose.

In summary, and without being bound by theory, the prolonged survivaland disease duration in fasudil treated SOD G93A mice as described inExample 1 may possibly be explained by an increased infiltration ofneuroprotective M2 microglial cells.

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The invention claimed is:
 1. A method of treating a subject suffering orprone to suffer from familial ALS, wherein the method comprisesadministering a pharmaceutical composition, comprising or consisting offasudil (1-(5-isoquinolinesulfonyl)homopiperazine), or a fasudilderivative selected from hydroxy-fasudil(1-(1-hydroxyl-5-isoquinolinesulfonyl)homopiperazine) anddimethyl-fasudil((S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine),or a pharmaceutically acceptable salt thereof, to the subject, whereinthe familial ALS is early stage ALS as diagnosable by the Awaji-shimadiagnostic criteria, and wherein the administration of said compositionis started presymptomatically.
 2. The method of claim 1, wherein thesubject is a female.
 3. The method of claim 1, wherein thepharmaceutical composition is to be administered orally.
 4. The methodof claim 1, wherein the pharmaceutical composition comprises or consistsof fasudil.
 5. The method of claim 4, wherein the fasudil is to beadministered to the subject in a dosage of 1-12 mg/kg body weight perday.
 6. The method of claim 1, wherein the pharmaceutical compositioncomprises or consists of a fasudil derivative selected fromhydroxy-fasudil and dimethyl-fasudil.
 7. The method of claim 6, whereinthe fasudil derivative is to be administered to the subject in a dosageof 10-1200 ng/kg body weight per day.
 8. The method of claim 1, whereinthe fasudil, fasudil derivative, or a pharmaceutically acceptable saltthereof is formulated as a formulation with sustained release orprolonged release.
 9. A method of improving motor coordination in asubject suffering or prone to suffer from familial ALS, wherein themethod comprises administering a pharmaceutical composition, comprisingor consisting of fasudil, or a fasudil derivative selected fromhydroxy-fasudil and dimethyl-fasudil, or a pharmaceutically acceptablesalt thereof, to the subject, wherein the familial ALS is early stageALS as diagnosable by the Awaji-shima diagnostic criteria, and whereinthe administration of said composition is started presymptomatically.10. The method of claim 9, wherein the subject is a female.
 11. Themethod of claim 9, wherein the pharmaceutical composition is to beadministered orally.
 12. The method of claim 9, wherein thepharmaceutical composition comprises or consists of fasudil.
 13. Themethod of claim 12, wherein the fasudil is to be administered to thesubject in a dosage of 1-12 mg/kg body weight per day.
 14. The method ofclaim 9, wherein the pharmaceutical composition comprises or consists ofa fasudil derivative selected from hydroxy-fasudil and dimethyl-fasudil.15. The method of claim 14, wherein the fasudil derivative is to beadministered to the subject in a dosage of 10-1200 ng/kg body weight perday.
 16. The method of claim 9, wherein the fasudil, fasudil derivative,or a pharmaceutically acceptable salt thereof is formulated as aformulation with sustained release or prolonged release.